The identification of the molecular connections between obesity, high fat diet and sedentary lifestyle and the promotion of insulin resistance, type 2 diabetes (T2D) and the metabolic syndrome is an area of intense investigation. Numerous studies from many labs indicate that potential pathogenic mediators converge at the level of Ser/Thr phosphorylation of the insulin receptor (IR) and/or its substrates (IRSs), including activation of insulin signaling itself (negative feedback), inflammatory cytokines and other mediators of inflammation (cross-talk), free fatty acids and cellular and oxidative stress. A large number of Ser/Thr kinases have been shown to lead to IR and/or IRS phosphorylation and by doing so potentially participate in mediating insulin resistance. However, most of these studies have utilized over-expression in cultured cells, and since most Ser/Thr kinases function in cascades, it has not been possible to determine primary vs. secondary effects. Specific mechanisms for inhibition are unknown if virtually every case. We are employing a proteomics approach to identify (1) discrete kinases that disrupt IP/IRS interactions and (2) sites of phosphorylation and mechanisms responsible for disruption of insulin signaling. Our approach utilizes a disruptive yeast tri-hybrid (Y3H) method specifically designed and developed for this purpose and mass spectrometry to identify discrete sites, the Y3H method and x-ray crystallography to determine mechanism, and analyses of rodent and patient tissue samples to assess potential relevance under physiological and pathological conditions. Specific aims include the use of (1) Y3H to screen a broad panel of Ser/Thr kinases to identify those that disrupt IR/IRS interactions, (2) mass spectrometry to identify specific sites of phosphorylation on IR and IRSs isolated from Y3H, (3) Y3H to identify and confirm specific sites of Ser/Thr phosphorylation and mechanisms of disruption of IR/IRS interactions, (4) newly developed phospho-specific antibodies to validate sites of phosphorylation in cells, animals and patient samples to determine physiological and pathophysiological conditions under which phosphorylation occurs, and (5) x-ray crystallography to visualize specific pathophysiological mechanisms for disruption of IR/IRS interactions. These studies should provide a growing framework for understanding major mechanisms of insulin resistance in obesity, T2D and the metabolic syndrome.